The outer surface of Gram-negative bacteria is covered with a remarkable, macromolecular glycolipid known as lipopolysaccharide (LPS), the hydrophobic anchor of which is lipid A. In Escherichia coli, lipid A consists of a hexa-acylated disaccharide of glucosamine with phosphate groups at the 1 and 4'positions. The minimal LPS substructure required for growth is lipid A with one or two additional sugars. Inhibition of any of the first seven enzymes that synthesize lipid A is lethal. These enzymes are expressed constitutively and are conserved in virtually all Gram-negative bacteria. Potent inhibitors have recently been discovered that target the second enzyme (LpxC) of pathway, with antibiotic activity comparable to ciprofloxacin. Lipid A (also known as endotoxin) is the active component of LPS that stimulates immune cells. During severe Gram-negative infections, the lipid A moiety of LPS, shed from bacteria, can cause excessive activation of macrophages and endothelial cells. Over-production of inflammatory mediators, such as TNF- alpha, IL-1beta, IL-6 and other proteins, damages small blood vessels. A full response to endotoxin leads to Gram- negative septic shock with multiple organ failure and death. A therapeutic approach has emerged with the discovery that certain lipid A-like molecules, including some precursors, are endotoxin antagonists. The signaling receptor for lipid A is the TLR4 protein, which is distantly related to the IL-1 receptor. TLR4 and its subunit MD2 on immune cells can discriminate between agonists and antagonists. One can re-engineer the lipid A of viable bacteria to be a TLR4 antagonist or a partial agonist, with potential applications in new vaccine development. In previous work, the P. I. identified the 9 constitutive and 10 inducible enzymes in E. coli and Salmonella, needed for lipid A biosynthesis and modification, respectively. The genes encoding these enzymes were also identified, primarily by expression cloning. The essential ABC transporter MsbA, which is closely related to the eukaryotic Mdr proteins, flips newly made LPS across the inner membrane, and is also required for LPS and phospholipid export to the outer membrane. The specific aims for the coming grant period are: I) the biochemical and genetic analysis of potent new LpxA and LpxC inhibitors;II) purification and characterization of the membrane enzymes LpxB, LpxK, KdtA, LpxL, and ArnT of lipid A biosynthesis;III) development of quantitative assays for the assembly of E. coli LPS core sugars and O-antigen;and IV) the design of in vitro systems for measuring LPS flip-flop and transport. These studies should accelerate the development of antibiotics that target the lipid A system and provide fundamental insights into the mechanisms of bacterial membrane assembly.